Method and Configuration for the Optical Detection of an Illuminated Specimen

ABSTRACT

A method and a configuration for the depth-resolved optical detection of a specimen, wherein a specimen or a part of the specimen is scanned by means of preferably linear illumination, the illumination of the specimen is periodically structured in the focus in at least one spatial direction, light coming from the specimen is detected and images of the specimen are generated, and at least one optical sectional image and/or one image with enhanced resolution is calculated through the specimen is calculated [sic], images are repeatedly acquired and sectional images are repeatedly blended while changing the orientation of the linear illumination relative to the specimen and/or spatial intervals between from lines exposed to detection light from the illuminated specimen region are generated for the line-by-line non-descanned detection on an area detector or a camera and/or, during a scan, light is further deflected upstream of the detector through the line in the direction of the scan of the specimen.

In microscopy, structured illumination is used for depth discriminationin the wide field [1] and for enhancing the resolution and the contrast[2]. Generally, a grating or another periodic structure is projectedinto the specimen [3] or an interference pattern is generated in thespecimen by means of interference of coherent component beams [4]. Byshifting the illumination structure, images are generated that differfrom one another with different phase angles of the period structure.Subsequently, these images are suitably blended with one another so asto obtain an optical sectional image and/or an image with enhancedcontrast and enhanced resolution. The disadvantage is that the signalfrom out-of-focus regions of the specimen is detected as well, which,because of the limited dynamic region of the detector, leads to areduced signal-to-noise ratio. The strength of the out-of-focus signallimits the useful sample thickness. This is of considerablesignificance, especially in cases in which the frequency of thestructure approaches the diffraction-limited threshold frequency of theoptical system, and the contrast of the structure is thereforenecessarily low. This invariably applies to cases in which the objectiveis enhancement of contrast and resolution.

One solution to this problem aims at a partially confocal detectionwhich is made possible by structuring a line of light and detecting thethereby excited fluorescent light by means of a slit detector [5].However, this method has a number of disadvantages. Structuring occursonly along the line. As a result, the effects of contrast and resolutionenhancement are limited to this direction. Thus, especially in cases ofnonlinear [6], but also linear, structures [7], the discrepancy betweenthe one direction with enhanced resolution and all other spatialdirections is significant. It is necessary to scan the line in a randomdirection in the specimen plane and to set the phase angle of the periodstructure. In the prior art, this requires separate actuators forcontrolling the relative phase angle and the scanning procedure.

It is the objective of the present invention to overcome the drawbacksof the prior art.

According to the present invention, the advantages of structuredillumination in the wide field (few optical components, highparallelization) are combined with the advantages of structuredillumination along a line (partially confocal suppression of thebackground signal for maximum contrast, high intensities in the focusfor nonlinear and linear specimen interactions). The proposedconfiguration males it possible to rotate the scanning directionrapidly, variably, and precisely, and to adjust the relative phase angleof the imaged structured periodic structure by means of only twoscanners. In addition, it enables a variably adjustable confocaldetection while allowing only very low losses of light in the detectionbeam path. In this context, reference is also made to DE 10155002 A1.

The solution according to the present invention is preferably aline-scanning microscope (see FIG. 1) with as few components in thedetection beam path as in a wide-field system. In concrete terms, thiscomprises an objective lens (27) which is corrected for an infinite beampath, the barrel lens (21), the main color divider (19), an emissionfilter (17) and the camera (15). In the excitation beam path, thebeam-shaping unit (8) which shapes the light beam of the light source(3), said light-beam having been intensity-modulated by the modulator(5) into a line that is modulated along the line width. In the practicalexample illustrated in FIG. 1, the beam-shaping unit comprises acombination of a line-shaping optics system (7) and a periodic structure(13), wherein (7) and (13) are combined to form a single mechanicalgroup (8) that can be rotated about the optical axis (1). By rotatingthe beam-shaping unit (8), which is preferably implemented by a rapidstepping motor, it is possible to set the orientation of the line imagedin the specimen x/y plane.

According to another embodiment of the present invention (not shown inFIG. 1), the beam-shaping unit (8) can also be implemented by means of asingle diffractive optical element which can also be rotated about theoptical axis (1). A diffractive element of this type can shape the linein one direction and structure the line in a direction orthogonalthereto in a single step.

Disposed further along the optical axis are a first scanner (9), asecond scanner (23) orthogonal to the first scanner (9), and a scanninglens (11). The axis of rotation or swivel axis (25) of the scanner (23)is disposed substantially orthogonal to the axis of rotation of thefirst scanner (9). The scanner (9) is used to shift the line in thespecimen in the x-direction, and the scanner (23) is used to shift theline in the y-direction.

Both scanners (9) and (23) are disposed near the conjugate pupil plane.

FIG. 1 shows the schematic assembly of the microscope according to thepresent invention. (1) is the optical axis, (3) is the light source, (5)is a switchable attenuator/AOM, (8) is a beam-shaping unit with aline-shaping optics system (7), for example, a cylindrical lens, (9) isscanner with an axis of rotation perpendicular to the drawing plane,(23) is a scanner with an axis of rotation (25) substantially parallelto the drawing plane, (11) is a scanning optics system, (13) is a maskwith a periodic structure in the intermediate image plane conjugate tothe specimen, (15) is a spatially resolved area sensor, e.g., a CCDreceiver matrix, (17) is an emission filter, (19) is a main colordivider, (21) is a barrel lens, (27) is a microscope lens, and (29) isthe specimen. The elements (7) and (13) are combined to form a singlemechanical group, the beam-shaping unit (8), which is preferablydisposed so as to be able to rotate about the optical axis (1).

Next, shifting the phase of the structured line and scanning the imagefield by means of the interaction of the two scanners (9) and (23) withthe AOM (5) will be described.

Without loss of generality, an example will be described, wherein theline in the specimen is oriented along the x-direction and scanning ofthe image field takes place in the y-direction, perpendicular to thex-direction. This also requires an orientation of the beam-shaping unit(8) to generate an orientation of the line in the x-direction.

During this line orientation, the scanner (23) serves to change thephase angle of the structure between two and more acquired images, whilethe scanner (9) is responsible for the scanning procedure in they-direction.

From the images acquired at different phase angles (“phase images”), asectional image is calculated (reconstructed). In this context,reference is made to DE 10155002 A1.

FIG. 2 shows the structure of the illumination.

If, during a linear scan by the scanner (9) over a time Δt=t3−t1, thecamera synchronously acquires an image with an exposure time of at leastΔt, the result obtained is equivalent to a wide-field image of thespecimen. At the same time, the out-of-focus background is detected aswell. According to the present invention, confocal filtering can be usedif the modulator (5), synchronously with the scanning procedure,periodically switches the illumination on and off in the y-direction aseach phase image that is needed to calculate a sectional image isacquired.

One advantage is that even during the switched-off intervals, thescanner, in addition to the continuous scanning motion with on and offswitching, can be rapidly moved to the next position with switched-onillumination in which the illuminated scanning procedure is continued.The scanner could also move step-by-step, similar to a stepping motor.

The method according to the present invention leads to an exposure inthe camera plane, which exposure is structured in the y-direction (seeFIG. 3). In one useful embodiment of the invention, the spatialintervals between the exposed lines of the camera are selected in such amanner that cross-talk of the out-of-focus background is minimizedduring the illumination of a line on the specimen into the region of thecamera that corresponds to the illumination of the next line in thespecimen. When the specimen is scanned according to the Nyquist theorem(one detector line corresponds to half the width of thediffraction-limited line), empirically a spatial interval of M=5 to 10lines between neighboring exposure lines should be sufficient. In thenext image acquired by the camera, the exposed line pattern ispreferably is shifted by one line, which is implemented by anappropriate delay in switching the modulator on. Thus, for example,first the 1st, 10th, 20th line and subsequently, the 2nd, 11th and 21stline are exposed.

This procedure of shifting the specimen illumination line by line isrepeated until all sections of the specimen in the image field have beenscanned, so that M images per phase angle are obtained as a result ofthis acquisition procedure.

As an alternative, to set the spatial interval between exposed lines,first, images could be acquired at several phase angles of the periodicstructure, and subsequently the spatial interval could be changed forthe acquisition of several more phase images.

In addition to the method already described above, each of these imagescan be created by repeatedly acquiring each image, preferably at thelowest possible intensity to spare the specimen, using the same scannersettings and then taking the mean. This method can reduce artifacts dueto bleaching phenomena taking place in the specimen. By blending Mimages per phase angle, it is now possible to adjust the confocality.

Specifically, it is necessary to subtract the exposed background betweenthe exposed receiver lines, which was detected by the receiver, for theindividual images. This background can be readily identified on thereceiver since the exposed lines in the specimen can be unambiguouslylinked to regions that are separated from one another on the camera.

If all M images of a phase angle are simply summed up, a resultcorresponding to the wide-field image is obtained. Summing up the imagesafter selection of the lines that correspond to the relevant illuminatedlines in the focus of the specimen leads to a confocal image. In thisstep, the image regions neighboring the selected lines are, asdescribed, masked and not analyzed. This corresponds to the function ofa virtual slit diaphragm, since the unused, masked image regionscorrespond to the detection sites of the out-of-focus scattered light.The confocality can be varied between 1 Airy unit (2 lines selected) andM Airy units (virtual slit diaphragm).

FIG. 3 shows the exposure pattern on the camera for confocal detectionwith the modulator on and off (AOM).

Compared to nonconfocal detection, the speed of image acquisition isdecreased by the factor M. Based on an image acquisition of 50 images/s,at M=5, a complete image can be obtained in 100 ms (at a phase angle ofthe structure). However, it should be noted that for each structureorientation, N=3 to 5 images at different phase angles must be acquired.Thus, in the case of a linear structure with 3 structure orientations,typically, 9 images must be taken [7], which, at M=5, leads to an imageacquisition time of approximately 1 s per plane.

A slightly more favorable situation results if the scanner (9) does notscan the image field uniformly (at speed v_(s)) but moves at a maximumspeed v_(max) during the times in which the laser is switched off.Although this makes higher demands on the control and synchronicity ofthe scanners, it increases the image acquisition time by the factor

${sf} = \frac{M}{{\left( {M - 1} \right)\frac{v_{s}}{v_{\max}}} + 1}$

i.e., approximately M-fold (if V_(max)>>v_(s)) or up to the maximumimage acquisition speed of the camera.

When linear structured illumination is used, it should be rememberedthat the length of the higher orders transmitted through the circularpupil is shortened as the structuring frequency increases (see FIG. 4),This means that the contrast of an interference with the 0th orderdecreases on the illumination side. However, if the passage through thepupil is symmetrical, the contrast of the interference between thehigher orders remains unchanged at 100%. In addition, the width of thediffraction-limited line in the image increases. At a structuringfrequency f that has been standardized to the threshold frequency, awidening b (line width divided by the minimum width at full numericalaperture NA) of

b=(2√{square root over (1−f ²)})⁻¹

results. For a typical structuring frequency of 90% of the thresholdfrequency (f=0.9), a widening of 15% results. At 95% of the thresholdfrequency, this widening increases to 60%. This widening does not havean influence on the resolution that is determined by the structuringfrequency and the transfer function of the objective lens, but it doesinfluence the suppression of the out-of-focus background and must betaken into consideration for confocal filtering.

FIG. 4 shows orders of the structured line illumination in the pupil(Fourier transformation of a structured line distribution).

A structured line is generated on the specimen by the interfering firstdiffraction orders. The spatial interval of the diffraction orders is s;a denotes the size of the pupil. The ratio between s and b is thestructuring frequency f that has been standardized to the thresholdfrequency.

The lines parallel to the x-direction seen in FIG. 3 represent only onestructure orientation. The orientation of the lines on the camera can beset by rotating the unit (8) (FIG. 1). The direction of shift and thephase angle of the periodic structure are set by means of the scanners(9) and (23).

FIG. 5 illustrates the general case with a random line orientation.

FIG. 5 explains how to set the scanning direction and the phase angle bymeans of the scanners with synchronous scanner movement (a) and scanningwith a scanner (b).

The phase angle (double arrow) of the projected structure is determinedby the relative constant offset of the two scanners (9) and (23) duringthe scan, while the direction of scan preferably perpendicular thereto(arrow) is defined by the relative speed of the two scanners. However,the specimen can also be scanned with scanner (9) only. This simplifiessystem control. Depending on the scan mode, it must be ensured that theimage field generally determined by the detector is illuminated ashomogeneously as possible even when the line is rotated.

In the configuration described so far, shaping of the structureprojected into the specimen is ensured by the beam-shaping unit (8).According to the invention, the unit (8) can be a combination of aline-shaping optics system (7) with a periodic structure (13). Theline-shaping optics system can comprise a Powell lens. The periodstructure can be a phase structure, an amplitude structure or acombination of the two. In addition, the entire beam-shaping unit (8)can be replaced with a diffractive optical element (see also DE 10155002A1). This element can generate one or more structured lines on thespecimen at a minimum spatial interval M in order to reduce the numberof shifts.

A potential problem of sequential scanning of the sample with M linepatterns arises when the specimen moves during the time of imageacquisition. This is a fundamental problem of the method for thestructured illumination and should be minimized as much as possible byminimal image acquisition times. This is why the sensitive detectionwith minimum fluorescence losses between the specimen and the detectoris so important. An alternative to sequential scanning with M linepatterns that makes it possible to acquire a single image instead of Mline images and yet allows confocal detection will be described below.To this end, one takes advantage of the fact that the line scanner scansthe specimen sequentially line-by-line. This makes it possible toimplement a discrete line-by-line deflection of the detection light byan additional element in the detection beam path so that a line patternas shown in FIG. 3 forms on the detector even though the specimen isscanned without spatial intervals. The prerequisite for depicting acomplete image on the detector in this manner is that this detector hasM more lines than are required for the image. A typical value is 500lines per image. At M=5, this leads to a required detector line numberof 2500. The element to be used to implement the line deflection couldbe, e.g., a galvanometer scanner upstream of the detector (see FIG. 6,scanner (24)). If the pixel size on the camera is 5 μm, the maximumangle of deflection is such that in the example mentioned above, anoffset of (2500−500)×5 μm=10 mm results on the camera. Assuming aspatial interval of 50 mm between the camera and the scanner, thiscorresponds to a scan angle of 5 degrees (for a deflection of 10degrees). To ensure that the line scanned in the specimen is not blurredacross the spatial intervals between the lines, it is useful to switchoff the exposure (e.g., by means of the AOM or AOTF) during the discretedeflection by scanner (24). However, a continuous exposure isconceivable as well. Since the scanning speed of the scanner (24) mustbe very high compared to that of scanner (9), the exposure can bedisregarded during the movement of the scanner (24). For example, thescanner (24) with the same axis of deflection as the scanner (9), whichas described above is responsible for the deflection in the y-direction,generates, for example, 10 offset discrete scan jumps within one lineposition of the sequential line scan of the scanner (9) before itadvances to the next detected line position. The scanner (9) can alsoscan continuously, while scanner (24) must always be operateddiscretely, with a high deflection speed. The time t_(j) between thescan jumps with a time t_(d) corresponds to the effective lineintegration time on the camera. At least M·t_(d)<t_(j) must apply.

On the area detector, these scan jumps generate spaced-apart signals ofthe illuminated specimen which approximately correspond to thespaced-apart regions of the detector as described in detail aboveespecially in connection with FIG. 3.

FIG. 6 shows the schematic assembly of the microscope with alternativedetection and an additional galvanometer scanner (24) with an axis ofrotation perpendicular to the drawing plane

When using optics systems with higher numerical apertures, such as arenormally used in microscopy, polarization must be taken intoconsideration if a structured illumination with the highest possiblecontrast of the structure in the specimen plane is to be obtained.Maximum contrast is possible only if the polarization of theilluminating light is perpendicular to the connecting line of thediffraction orders in the pupil plane (i.e., perpendicular to theposition of the line in an image plane), as shown in FIG. 4. Thepolarization of the illuminating light must therefore be rotated withthe rotation of the beam-shaping unit (8) synchronously with therotation of the diaphragm. The former [sic] can preferably be generatedby rotating a λ/2 plate in the beam path of the linearly polarizedexcitation light, with the angle of rotation of the wave plate beinghalf as large as that of the beam-shaping unit. Accordingly, a rotatablewave plate should be disposed in

the beam path of FIGS. 1 and 6 between the source (3) and the main colordivider (19). As an alternative, the beam-shaping unit can also befitted with a polarizer that transmits only correctly oriented, linearlypolarized light. This entails a rotation-dependent loss of light, seeFIG. 7, which can be compensated for by suitably synchronized lightmodulation.

FIG. 7 shows the loss of light as a result of the rotation of apolarizer in combination with the beam-shaping unit (8) and compensationof the loss by adjusting the power

The invention is not limited to the embodiments described above.

Within the context of the actions and knowledge of those skilled in theart, modifications and changes can be covered by the inventive thoughts,

For example, the present invention can be applied analogously to otherillumination distributions, such as multi-point configurations (U.S.Pat. No. 6,028,306) and other point configurations, including Nipkowdisks, and to detection in wide field.

REFERENCES

-   [1] Nell M. A. A., Juskaltis, R., Wilson T.; “Method of obtaining    optical sectioning by using structured light in a conventional    microscope”, Opt. Lett. 22(24): 1905-1907, 1997-   [2] Lukosz W., Marchand M., “Optische Aufiösung unter Überchreitung    der beugungsbedingten Aufiösungsgrenze”, Optica Acta 16, 241-255,    1963-   [3] Heintzmann R., Cremer C., “Laterally Modulated excitation    microscopy: Improvement of resolution by using a diffraction    grating”, in Proc. of SPIE 3568: 185-196, 1998-   [4] Neil M. A. A. Juskaltis, A., Wilson, T., “Real time 3D    fluorescence microscopy by two beam interference illumination”, Opt.    Comm. 153: 1-4, 1998-   [5] U.S. Pat. No. 6,947,127 B2, 2005-   [6] Heintzmann R., Jovin T. M., Cremer C., “Saturated patterned    excitation microscopy—a concept for optical resolution improvement”    JOSA A, 19(8): 1599-1609, 2002-   [7] Gustafsson, M. G. L., Agard, D. A., Sedat, J. W., “Doubling the    lateral resolution of wide-field fluorescence microscopy by    structured illumination”, in Proc. of SPIE 3919: 141-150, 2000-   [8] Gustafsson M. G. L. “Nonlinear structured-illumination    microscopy: wide-field fluorescene imaging with theoretically    unlimited resolution”, PNAS 102: 13081-13086, 2005

1. A method for the depth-resolved optical detection of a specimen,wherein a specimen or a part of the specimen is scanned by means oflinear illumination, the illumination of the specimen is periodicallystructured in the focus in at least one spatial direction, light comingfrom the specimen is detected and images of the specimen are generated,and at least one optical sectional image and/or one image with enhancedresolution is calculated through the specimen is calculated [sic],characterized in that images are repeatedly acquired and sectionalimages are repeatedly blended while changing the orientation of thelinear illumination relative to the specimen.
 2. The method as in one ofthe preceding claims [sic], wherein the line is rotated about theoptical axis and the images are generated and the sectional images areblended at different angles of rotation.
 3. The method as in one of thepreceding claims, wherein, to generate lines, a beam-shaping unit isjointly rotated with means for structuring the illuminating light.
 4. Amethod for the depth-resolved optical detection of a specimen, wherein aspecimen or a part of the specimen is scanned by means of preferablylinear illumination, the illumination of the specimen is periodicallystructured in the focus in at least one spatial direction, light comingfrom the specimen is detected and images of the specimen are generated,and at least one optical sectional image and/or one image with enhancedresolution is calculated through the specimen is calculated [sic], inparticular as in one of the preceding claims, characterized in thatspatial intervals between lines exposed to detection light from theilluminated specimen region are generated for the line-by-linenon-descanned detection on an area detector or a camera.
 5. The methodas in one of the preceding claims, wherein during the preferably linearillumination and detection, the illumination is repeatedly switched onand off.
 6. The method as in one of the preceding claims, wherein duringthe scanning of the specimen, the light is repeatedly interrupted sothat a spatial interval is formed between two illuminated specimenregions.
 7. The method as in one of the preceding claims for theconfocal generation of images, wherein an image is calculated bypartially or completely masking the spatial intervals between cameraregions associated with the exposed specimen regions and the images thusobtained are blended.
 8. The method as in claim 7, wherein the imagesare blended in such a manner that neighboring scanned regions of thespecimen are properly scaled and adjacently aligned in the blendedimage.
 9. A method for the depth-resolved optical detection of aspecimen, wherein a specimen or a part of a specimen is scanned by meansof preferably linear illumination, the illumination of the specimen isperiodically structured in the focus in at least one spatial direction,light coming from the specimen is detected and images of the specimenare generated, and at least one optical sectional image and/or one imagewith enhanced resolution is calculated through the specimen iscalculated [sic], in particular as in one of the preceding claims,wherein, during a scanning procedure, light is further deflectedupstream of the detector through the line in the direction of the scanof the specimen.
 10. The method as in one of the preceding claims,wherein the speed of the light deflection is greater than the speed ofthe relative movement between the specimen and the illuminating light.11. The method as in one of the preceding claims, wherein the light isdeflected step-by-step.
 12. The method as in one of the precedingclaims, wherein the light is deflected continuously.
 13. The method asin one of the preceding claims, wherein, during a rotation of theilluminating line, the polarization of the illuminating light issynchronously rotated with the rotation.
 14. The method as in one of thepreceding claims, wherein repeated scanning takes place and the positionof the periodic structure on the specimen and/or the position of theilluminating light on the specimen is shifted.
 15. The method as in oneof the preceding claims, wherein several images with different imagephases are acquired and sectional images are calculated therefrom. 16.The method as in one of the preceding claims, wherein the images areacquired with different image phases with a constant spatial intervalbetween illuminated/detected sections.
 17. The method as in one of thepreceding claims, wherein the position of the spatial interval ischanged and several images with different image phases are acquired foreach position and sectional images are calculated therefrom.
 18. Themethod as in one of the preceding claims, wherein first the position ofthe spatial interval for one position of the structure is changed andspecimen images are acquired, and subsequently this procedure isrepeated for the next position of the structure.
 19. The method as inone of the preceding claims, wherein the position of the spatialinterval is changed in such a manner that substantially all specimenregions are sequentially illuminated line-by-line and the specimen lightis detected.
 20. The method as in one of the preceding claims, whereinthe light is interrupted by decreasing the intensity by means of anelectro-optical and/or acousto-optical modulator.
 21. The method as inone of the preceding claims, wherein, for the purpose of the periodicstructuring of the illumination, a light beam is divided into severalcomponent light beams, which light beams are interferometricallyoverlapped and shaped into a line.
 22. The method as in one of thepreceding claims, wherein the light resulting from a nonlinearinteraction of the illumination with the specimen or a part of thespecimen . . . [word or words missing] and is detected.
 23. The methodas in one of the preceding claims, characterized in that linear scanningis carried out simultaneously with several lines.
 24. The method as inone of the preceding claims, characterized in that the optical sectionthickness or optical resolution is varied as structures with differentmodulation frequencies are imaged.
 25. The method as in one of thepreceding claims, characterized in that during illumination with severalwavelengths, the section thickness is identically set by adjusting eachmodulation frequency.
 26. A configuration for the depth-resolved opticaldetection of a specimen, comprising means for the preferably linearillumination of the specimen with at least one wavelength, means forspatially structuring the illuminating light in at least one plane,means for generating a relative movement between the specimen and theilluminating light, means for imaging the light influenced by thespecimen on at least one detector, and means for calculating at leastone optical sectional image and/or one image with enhanced resolutionfrom the spatial information of the light influenced by the specimen,characterized in that means for changing the orientation of the linearillumination relative to the specimen are provided.
 27. Theconfiguration as in claim 26, wherein a jointly rotatable unitcomprising a beam-shaping unit to generate lines and means forstructuring the illuminating light in the beam path is provided.
 28. Aconfiguration for the depth-resolved optical detection of a specimen,comprising means for the preferably linear illumination of the specimenwith at least one wavelength, means for spatially structuring theilluminating light in at least one plane, means for generating arelative movement between the specimen and the illuminating light, meansfor imaging the light influenced by the specimen on at least onedetector, and means for calculating at least one optical sectional imageand/or one image with enhanced resolution from the spatial informationof the light influenced by the specimen, in particular as in one of thepreceding claims, wherein an area detector or a camera for thenon-descanned detection of the specimen light is provided and whereinmeans for interrupting the light during the scan are provided so as togenerate a spatial interval between illuminated specimen regions and/orto generate spatial intervals between lines exposed with detection linefrom the illuminated specimen region on the area detector.
 29. Theconfiguration as in claim 28, wherein intensity control means aredisposed in the illuminating beam path.
 30. The configuration as inclaim 28 or 29, wherein an electro- or acousto-optical modulator forlight interruption is provided.
 31. A configuration for thedepth-resolved optical detection of a specimen, comprising means for thepreferably linear illumination of the specimen with at least onewavelength, means for spatially structuring the illuminating light in atleast one plane, means for generating a relative movement between thespecimen and the illuminating light, means for imaging the lightinfluenced by the specimen on at least one detector, and means forcalculating at least one optical sectional image and/or one image withenhanced resolution from the spatial information of the light influencedby the specimen, in particular as in one of the preceding claims,wherein a scanner is disposed in the detection beam path in order toexpand the specimen light discretely on the detector or continuously onthe detector during the line-by-line scan.
 32. The configuration as inone of the preceding claims, characterized in that at least one scanneris provided as a means for generating the relative movement.
 33. Theconfiguration as in one of the preceding claims, characterized in thatthe means for structuring the illumination is an optical element that ispreferably rotatable about the optical axis and that is structuredrelative to its transparency.
 34. The configuration as in one of thepreceding claims [sic], characterized in that, in order to set differentimage phases of the structure, the position of at least one scanner canbe adjusted.
 35. The configuration as in one of the preceding claims,characterized in that, in order to set different frequency structures,gratings of different periodicities that can be rotated into the beampath are provided.
 36. The configuration or the method as in one of thepreceding claims, in a microscope, preferably in a laser scanningmicroscope.